The existence of lipoprotein lipase (hereinafter referred to an "LPL") as a clearing factor was recognized in 1943 by P. F. Hahn through his study on the circulatory amount of red blood cells. P. F. Hahn found that the injection of heparin to an alimentary lipemic dog results in clearing of the milky plasma. Thereafter, it was elucidated that the clearing mechanism is due to the hydrolysis of lipoprotein with LPL. LPL had been detected in the tissues of various animals. Thus, LPL plays an important role in lipid metabolism in animals.
Arima et al, Agr. Biol. Chem., Vol. 30, p. 515 (1966) reported that an enzyme similar to LPL of animal origin exists in microorganisms and this enzyme has been called microbial LPL. Because the microbial LPL can be produced in large quantity, studies on the utilization of microbial LPL have been promoted. In particular, applications regarding the quantitative determination of triglycerides in blood have been developed.
Microorganisms capable of producing LPL include various genera such as Pseudomonas, Mucor, Streptomyces, Serratia, Aeromonnas, Bacillus (see Agr. Biol. Chem., Vol. 31, p. 924 (1967), JP-B-41-7836, and JP-B-58-37835 (the term "JP-B" as used herein means an "examined published Japanese patent application"), and Rhizopus (see Agr. Biol. Chem., Vol. 43, p. 2125 (1979), and JP-B-58-37834). However, since all of these microorganisms are mesophiles, LPL produced therefrom exhibits poor stability.
With the recently increasing use of enzyme assays in clinical examinations, the unstability of enzymes has given rise to great problems. Techniques for obtaining thermostable enzymes from thermophilic microorganisms, have been developed to improve enzyme stability.
However, thermostable LPL has not yet been developed and has thus been keenly demanded.
Further, in cases when LPL is used in the quantitative determination of triglycerides in blood, LPL must exhibit high activity to form glycerol so as to reduce the requisite amount of the reagent used and to shorten the reaction time required. Thus, the use of any known LPL is not satisfactory.
It is known that lipases inclusive of LPL exhibit specificity to the three ester linkages of a triglyceride substrate. In the quantitative determination of blood triglyceride using LPL, glycerol formed by hydrolysis is preferably introduced into a detection system by a coupling enzyme. In order to form glycerol in an amount proportional to the triglyceride amount, it is preferable to use an LPL capable of hydrolyzing the three ester linkages without showing selectivity. The positional specificity of known LPL to ester linkages, being expressed in a percentage of glycerol formation activity to fatty acid formation activity, is generally low. The highest of the cases reported so far, is 1.97% of LPL originating in a microorganism belonging to the genus Pseudomonas as described in LP-A-59-187780 (the term "JP-A" as used herein means an "unexamined published Japanese patent application").